Polarized exocytosis is fundamental to a wide range of biological processes such as the release of neurotransmitters and the generation of epithelial basolateral asymmetry. Exocytosis occurs when post-Golgi secretory vesicles are tethered to and fuse with the plasma membrane (PM). Vesicle tethering is mediated by the exocyst, an evolutionarily conserved octameric protein complex. The exocyst component Exo70 physically interacts with phospholipids and is localized to the bud tip in the budding yeast Saccharomyces cerevisiae. Exo70 may act as a "landmark" protein to target vesicles to the PM. The spatial and temporal regulation of vesicle tethering controls the location and the kinetics of exocytosis;therefore, studying the regulation of Exo70 should be important for the understanding of the molecular basis of polarized exocytosis and cell growth. Aim 1: Determine the role of Gic1 and Gic2 on Exo70 function at the PM. The small GTPase Cdc42 is a regulator of polarized exocytosis. Cdc42 controls localization of Exo70 at the bud tip, but not through direct binding, suggesting that it controls Exo70 through downstream effector(s). Gic1 and Gic2, effectors of Cdc42, are localized to the bud tip during early budding stages in yeast and are important for the establishment of cell polarity. The interactions between Gic1/2 with the exocyst components (specifically Exo70), and their role in secretion and Exo70 polarization will be examined through genetic, biochemical, and cell biological methods. Aim 2: Identify and characterize novel regulators of Exo70 function. Recent studies indicate that Exo70 functions in parallel with another exocyst component, Sec3, in vesicle tethering. While mutations in either Sec3 or Exo70 have little effect on secretion and cell growth, mutations in both genes result in cell lethality. In order to identify additional regulators of exocytosis, yeast cells expressing mutant Sec3 will undergo random mutagenesis. If the mutation occurs in Exo70 or an Exo70 regulator, its combination with the Sec3 mutation should lead to cell growth defect or inviability. Experiments will be performed to identify these proteins and characterize their roles in polarized exocytosis. Relevance to Public Health: Exocytosis is a basic biological process conserved from yeast to mammals;it controls release of factors such as hormones and neurotransmitters, and incorporation of membrane for cell growth and migration. Defects in the timing and/or location of exocytosis may play a role in diseases such as cancer, diabetes and polycystic kidney disease. This study will focus on potential regulators of exocytosis in order to better understand the spatial and temporal control of exocytosis.